Process for determining a resistance to activated protein C

ABSTRACT

A process for determining a resistance to activated protein C of a test specimen of human plasma following the steps of: (1) mixing together (a) the test specimen of human plasma, (b) a reactant deficient in factor V which supplies at least most of the coagulation factors other than factor V, and (c) the venom of  Crotalus viridis helleri  which specifically activates factor X to Xa, and incubating the mixture of (a), (b) and (c) for at least one minute at a temperature of between 10 and 45° C.; (2) introducing into the incubated mixture(i) Ca 2+  or (ii) Ca 2+ +exogenic activated protein C; and (3) determining the coagulation time (i) in the absence of activated protein C and (ii) in the presence of activated protein C. Steps (1) to (3) are repeated, but replacing, in step (1), the test specimen with a normal plasma as control and correlating resistance to activated protein C by comparing the determinations made in steps for the test specimen and for the normal plasma. The initiation of coagulation is caused by activating factor X to Xa using the venom of  Crotalus viridis helleri  in the presence of (i) Ca 2+  or (ii) Ca 2+ + exogenic activated protein C.

This application is a 371 of PCT/FR 98/01148 filed Jun. 5, 1998 whichclaims priority to France 97 07 041 filed Jun. 6, 1997.

FIELD OF THE INVENTION

The present invention relates to a new use of a particular snake venom,the venom of Crotalus viridis helleri (this venom is referred tohereinafter as CVH), in the field of determining the reactivity of theactivated protein C, this determination including that of the activityof the functional protein C and that of resistance to the activatedprotein C (activated protein C is called APC herein and resistance toAPC is abbreviated as APC-R; the other abbreviations used are definedfurther below). The invention likewise relates to a method fordetermining APC-R, using CVH and a dosing kit enabling this method to beused.

PRIOR ART

The study of hereditary thromobophiliacs has shown that APC-R is mainlyassociated with a mutation on the gene coding for factor V. Thismutation, called “Leiden's mutation,” “mutation R506Q” or “mutation⁵⁰⁶R→⁵⁰⁶Q,” appears in the sequence of the amino acids of human factor Vby the replacement of Arg by Gln in position 506, and favors theappearance of thromboses, often venous (in the lower body members with,in some cases, pulmonary embolism). See particularly in this connectionthe publications of R. M. Bertina et al., Nature, 1994, 369, pages64-68, of M. Kalafatis et al., J. Biol. Chem. 1994, 269, pages31869-31880, of H. De Ronde et al., Thromb. Haemost., 1994, 72, pages880-886, of P. J. Svensson et al., N. Engl. J. Med., 1994, 330, pages517-522, of B. Dahlbäck et al., Proc. Natl. Acad. Sci. USA, 1993, 90,pages 1004-1008, and of B. Dahlbäck et al., Proc. Natl. Acad. Sci. USA,1994, 91, pages 1396-1400.

Other factor V anomalies can be at the origin of APC-R. In particular,it is possible that an untimely cleavage in position 306 (i.e., ³⁰⁶Arg)of the amino acid sequence of factor V and/or Va is one of the otherpossible causes of APC-R.

The determination of APC-R was employed initially (particularly by anAPTT, KCCT or other such test) to detect the existence of a thromboticdisorder by comparing the coagulation time, which is prolonged by theaddition of APC, of a control plasma with one that is shorter in case ofan irregularity in relation to the preceding one, of a sample of humanplasma to be tested with APC added.

See in this connection: C. A. Mitchell et al., N. Engl. J. Med., 1987,317, pages 1638-1642, L. Amer et al., Thromb. Res., 1990, 57, pages247-258, and B. Dahlbäck et al., Thromb. Haemost., 1991, 65, Abstract39, page 658.

The only kit presently available on the market (it is sold by the nameof “Coatest APC-Resistance” by the firm of Chromogenix) uses the processdescribed in the disclosure of B. Dahlbäck et al., Thromb. Haemost.,1991, 65, Abstract 39, page 658, referred to above and explained inWO-A-9310261, and the article by B. Dahlbäck et al., Proc. Natl. Acad.Sci. USA, 1993, 90, pages 1004-1008, referred to above. This processcomprises mixing one volume of the plasma to be tested or of the controlplasma with one volume of the APTT reactant [PL +surface activator(silica, kaolin or glass)], incubation for 4 minutes at 37° C., thenstarting coagulation by means of one volume of a solution of CaCl₂, onthe one hand, or of one volume of a solution of CaCl₂ and APC, on theother.

This process is unsatisfactory in that, being sensitive particularly tothe presence of factor VIII, heparin, PS, CAC's and VKA's, it gives toomany false negative and false positive results. Improvements havetherefore been proposed in WO-A-9615457, WO-A-9604560 and EP-A-0711838.

WO-A-9615457 proposes an APC-R determination in which the plasma to betested is placed in contact with a procoagulant reactant (tissue factorin this case) and a VdfP; then, after incubation, coagulation is startedwith CaCl₂ or CaCl₂+APC, the variation of the coagulation time beingthen determined by comparison with a control plasma.

WO-A-9604560 aims at an improved technique for detecting APC-R. Thistechnique is based on the use of an exogenic reactant that specificallyactivates the V factor to Va, to supplement an activation of thecoagulation either by the transformation X→Xa, or by the transformationof prothrombin to thrombin by a mechanism depending on the V factor.

WO-A-9604560 recommends particularly,

as an exogenic reactant, a snake venom such as the venom of Naja niveato activate V to Va, and

for the supplemental activation: either RVV-X to activate X to Xa,either a snake venom (or venom extract), such as the venoms ofPseudonaja textilis, Notechis scutatus or Oxyurnus scutellatus, for theactivation of prothrombin to thrombin by a mechanism depending on factorV.

For the determination of APC-R, EP-A-0711838 recommends a processcomprising;

(a) the mixing of a plasma to be tested with a reactant A with a lowcontent of factor V, particularly VdfP,

(b) the addition of a reactant B that directly or indirectly activatesfactor V to Va, particularly a viper venom such as RVV (with by itsRVV-V fraction induces the activation of V to Va and which by its RVV-Xfraction induces the activation of X to Xa) or the venom of Echiscarinatus (which induces the activation of prothrombin to thrombin andthen activates V to Va indirectly).

c) the addition of a reactant C permitting degradation of the Va factor,viz., APC [(or the mixture PC +activator (particularly ACC)], and

d) the addition of reactants permitting the determination of theresidual activity of the Va factor, on the condition that the proportionof the volume of the plasma sample to be tested with respect to thetotal volume is no more than 20%, advantageously less than or equal to10% (“the content of the sample volume being a maximum of 20%, butpreferably less than or equal to 10%,” according to claim 1, page 13,lines 39-40 of EP-A-0711838).

This process has the disadvantage of calling for volumes of plasma (tobe tested, or plasma for control), of reactant A and of reactant B whichare all three different. This circumstance does not make it easy tomeasure the coagulation time with a fully automatic apparatus.

The aforesaid processes of WO-A-9604560 and EP-A-0711838 make itpossible to relieve most of the disadvantages of the “CoatestAPC-Resistance.” However, these two documents neither describe norsuggest the use of the particular snake venom of the invention, viz.CVH, to initiate coagulation at the level of the X factor by thetransformation X→Xa.

The only effective technique today for determining APC-R is based onmolecular biology (MB). It is lengthy, fussy, burdensome and calls forprepared material and highly qualified personnel. Moreover, itsemployment must satisfy Article L. 145-15 of French Law No. 94-653 ofJul. 29, 1994 which requires obtaining the written consent of thepersons for whom a genetic study is undertaken.

THE SUMMARY OF THE INVENTION

It is intended to provide a new technical solution for the determinationof APC-R which (i) will be simple at the level of practice, (ii) will beat least as effective as those considered in the above-mentioneddocuments WO-A-9604560 and EP-A-0711838, and (iii) is based oninitiating coagulation at the X factor (i.e., transformation X-Xa) whoseimportance has been recognized in WO-A-9604560 (see page 5, lines 1-12).

The aim is to improve sensitivity by reducing the number of falsepositive and false negative results. With this in mind it is proposed toemploy an APC-R determination process by classical mechanisms, but withthe peculiarity that a new reactant or means is used to initiatecoagulation by the transformation X-Xa, this means or reactant improvingsensitivity.

The new technical solution according to the invention is based on theselection of a particular activator to initiate coagulation at the Xfactor (i.e., X→Xa). This particular activator is a snake venom, namelyCVH.

According to a first aspect of the invention, a new use for a snakevenom is announced, for starting coagulation by activating the X factorto Xa for the purpose of determining the reactivity of the activatedprotein C, particularly the functional activity of the protein C (PC) ofa human plasma being tested, the said use being characterized in that itresorts to the venom of Crotalus viridis helleri as the coagulationinitiating substance, in the presence of Ca²+, at the X factor, the saidvenom activating X selectively to Xa without being influenced by thepresence or absence of other coagulation factors.

According to a second aspect of the invention, a new method ofdetermining the APC-R of a test specimen of human plasma, the method,which employs the initiation of coagulation at the X factor by means ofa snake venom in the presence (i) of Ca²⁺ or (ii) a mixture of Ca²⁺ andexogenic APC, then the evaluation of the coagulation time, (i) in theabsence of APC and (ii) in the presence of APC, respectively, incomparison with a normal plasma, being characterized by comprising stepsconsisting of

(1) bringing into contact

(a) a specimen of the human plasma to be tested,

(b) a reactant deficient in V factor (VdfR) as a product furnishing most(preferably all) of the coagulation factors other than V factor, and

(c) CVH as a product specifically activating X to Xa, and incubating themixture thus obtained for at least 1 minute at a temperature between 10and 45° C., preferably for 3 to 5 minutes at a temperature of 35 to 40°C. and, better, for 4 minutes at 37° C.;

(2) introducing into the mixture thus incubated (i) Ca²+or (ii)Ca²⁺+exogenic APC, respectively;

(3) evaluating the coagulation time (i) in the absence of APC and (ii)in the presence of APC, respectively;

(4) repeating steps (1) to (3) by replacing, in step (1), the testspecimen of human plasma with a normal plasma as control, and

(5) estimating APC-R by comparing the evaluations obtained in steps (3)and (4).

According to a third aspect of the invention, a variant of this processis offered, which can be performed directly without the need, on the onehand, to make a comparison with a control plasma (normal plasma or poolof normal plasmas) and, on the other hand, to perform a measurementwithout the addition of APC. This variant comprises steps (1), (2, ii)and (3, ii) of the principal process, on the one hand, then theestimation of APC-R by comparing the coagulation time (T) obtained instep (3, ii) to an experimentally determined reference coagulation time(To) (having a value of approximately 110 s on a statistical basis), onthe other hand,

T<To indicating generally that the test specimen of human plasma has aresistance to activated C protein, and

T>To indicating generally that the test specimen of human plasma has noresistance to activated C protein.

This variant is advantageous in that the determination of APC-R issimpler, less expensive and easier to automate than the main processconsidered above.

According to another aspect of the invention, a dosage kit is offeredwhich includes:

a first reactant which is a plasma deficient in V factor,

a second reactant constituted by CVH, and

a third reactant constituted by APC in a calcium medium.

ABBREVIATIONS

For convenience, the following abbreviations and acronyms have been usedin the present description.

ACC Venom of Agkistrodon contortrix; this venom is a product whichselectively activates PC to APC

APC Activated protein C

APC-R Resistance to activated protein C (when “plasma APC-R” isinvolved, it is understood that the plasma is resistant to APC).

APTT Activated partial thromboplastin time

ATIII Antithrombin III

CAC Circulating anticoagulant

CVH Venom of Crotalus viridis helleri

KCCT Kaolin-cephalin clotting time

MB Molecular biology test, here employing a coding nucleic probe for apeptide fragment of about ten amino acids comprising ⁵⁰⁶Q, and thedetection of the mutation by hybridization of the probe afteramplification (by PCR) of the corresponding region of the DNA

MB+ Identifies a plasma in which the V factor comprises the mutationR506Q (i.e., plasma “positive” according to the MB test)

MB− Identifies a plasma in which the V factor does not comprise themutation R506Q (i.e., plasma “negative” according to the MB test)

OKB Owren-Koller buffer; this is a veronal buffer

PB Prionex buffer (buffer containing the polypeptide fraction with porkcollagen removed)

PC Protein C

PCR Polymerase chain reaction

PL Phospholipids

PS Protein S

RVV Russel viper venom (Vipera russeli); this venom activates factor Vto Va and factor X to Xa

RVV-V Purified fraction of RVV activating factor V to Va

RVV-X Purified fraction of RVV activating factor X to Xa

VdfP Plasma deficient in factor V

VdfR Reactant deficient in factor V

VKA Vitamin K antagonist

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the variation of the normalized ratio N-APC-R-SR, which isdefined further below and represented on the ordinates in relation tothe concentration (% v/v) of the plasma (control pool or APC-R plasma)in a mixture of plasma+VdfP, this concentration being represented on theabscissae; and

FIG. 2 represents the variation of the said normalized ratio N-APC-R-SR(on the ordinates) with the concentration (% v/v) of an APC-R plasma ina mixture of APC-R plasma plus plasma control pool (on the abscissae).

DETAILED DESCRIPTION OF THE INVENTION

The use of the particular venom CVH according to the invention makes itpossible to know the reactivity of APC, and in particular the functionalactivity of PC on the one hand and APC-R on the other. Of course, thedetermination of APC-R is more important at the diagnostic level thanthat of the functional activity of PC. Consequently, the determinationof the functional activity PC is only secondary in relation to thedetermination of APC-R which makes it possible to estimate the risk ofthrombosis in the patient being tested.

CVH is a commercial product available on the market. It can be obtainedin purified and lyophilized form from the Sigma company. Prior to theinvention this venom was used mainly for the production of an antiserumagainst snakebite of Crotalus viridis helleri. In practice, lyophilizedCVH will be diluted with PB to a concentration of 0.1 to 0.15 μg/ml(preferably a concentration of 0.12 μg/ml, which corresponds to an endconcentration, in the employment of the process, of 0.03 μg/ml).

The reactant VdfR is chiefly a plasma VdfP. The expression “deficient infactor V” signifies a reactant (VdfR) or a plasma (VdfP) which is eitheressentially of reduced factor V content or contains no factor V. VdfR orVdfP are used according to the invention as a substrate plasma which canbe of synthetic, animal or human origin. Advantageously, a VdfPcontaining no factor V will be employed, and will be obtained byimmunodepletion. To prevent any interference from theanti-phospholipids, particularly those of lupus, it is recommended thatVdfR or VdfP be enriched with PL.

According to one particular embodiment, in step (1) a test specimen ofhuman plasma obtained by treatment with trisodium citrate (when it wastaken) is used, centrifuged at 3500 g for about 15 minutes, and dilutedwith OKB. The normal plasma used as control in step (4) is obtained inthe same way.

In a practical way, bearing in mind the notations APC-R-SR andN-APC-R-SR proposed in the aforementioned article of H. De Ronde et al.,in step (3) and in step (4) sensitivity to APC is evaluated for the testspecimen of human plasma and for the control plasma, the sensitivitybeing the ratio (called APC-R-SR) of the coagulation time in thepresence of APC to the coagulation time without APC:$\text{APC-R-SR} = \frac{{coagulation}\quad {time}\quad {in}\quad {presence}\quad {of}\quad {APC}}{{coagulation}\quad {time}\quad {in}\quad {the}\quad {absence}\quad {of}\quad {APC}}$

and in that in step (5) APC-R is reckoned by the normalized ratio(called N-APC-R-SR) of the sensitivity to APC of the test specimen ofhuman plasma to the APC sensitivity of the normal control plasma:$\text{N-APC-R-SR} = \frac{\left( \text{APC-R-SR} \right)\quad {specimen}}{\left( \text{APC-R-SR} \right)\quad {control}\quad {plasma}}$

In the process of the invention, a value of the normalized ratioN-APC-R-SR greater than 0.80 indicates that the human test specimengenerally offers no resistance to the activated protein C, and a valueof the normalized ratio N-APC-R-SR less than 0.80 indicates that thetest specimen generally does offer resistance to the activated proteinC. The normal plasma used as control is advantageously a pool of normalhuman plasmas.

When the value of the normalized ratio is within the gray area(0.72≦N-APC-R-SR≦0.90) for a concentration x (% v/v) of a plasmaspecimen in a mixture of x (plasma) +y (VdfP) or x +y=100%, it isrecommended to increase the concentration of the plasma, that is,inversely, to reduce the concentration of VdfP in the said mixture x+y.Thus a value of N-APC-R-SR is obtained which is outside of the gray area(see FIG. 1 below).

Advantageously, identical volumes of each of the reactants will be usedaccording to the invention, viz.: the human test plasma (preferablydiluted 1:10 in OKB), VdfR (or VdfP), CaCl₂ solution, CaCl₂+APCsolution, control plasma (or control plasma pool), and solution (ordilution) of CVH (preferably CVH in water containing 1% v/v of PB).

The evaluation of APC-R in steps (3), (4) and (5) is of a type known inthe prior art, which corresponds to the rate of conversion of asubstrate. The coagulation time is measured either directly(particularly by means of a coagulometer), recording the instantcorresponding to the thrombin formation generated in the reactingsystem, the substrate being then prothrombin. The substrate preferredaccording to the invention is VdfP.

The dosage kit or set according to the invention will advantageouslyinclude a VdfP enriched with PL. The kit can also contain CaCl₂ forpreparing the aqueous solution of CaCl₂ at the concentration required inorder to supply the Ca²+ions that induce coagulation. The timer isstarted when the Ca²⁺ ions are introduced.

According to the preferred embodiment of the process of the invention:

in step (1),

(A) the human blood test specimen, diluted with OKB to a concentrationequal to or more than 1/20 v/v, preferably to a concentration of 1/10v/v.

(B) a human plasma deficient in factor V and enriched with PL, as thereactant VdfR, and

(C) the venom of Crotalus viridis helleri are brought into contact andincubated for 4 minutes at 37° C.;

in step (2), into the mixture thus incubated, (i) Ca²⁺ or (ii) themixture of Ca²⁺ plus exogenic APC are placed;

in step (3) the APC sensitivity of the test human plasma to APC isevaluated;

in step (4), all of steps (1) to (3) where, in step (1), the testspecimen of human plasma was replaced with a normal human plasma, so asto evaluate the APC sensitivity of the normal human plasma, and

in step (5), the APC-R is estimated by the normalized ratio n-APC-R-SR ,and a value of the ratio greater than 0.80 (preferably, N-APC-R-SR>0.90)indicates that the test specimen of human plasma has no resistance tothe activated protein C, and a value of the ratio n-APC-R-SR of lessthan 0.80 (preferably N-APC-R-SR<0.72) indicates that the test specimenof human plasma does offer resistance to the activated protein C, thenormal plasma used as control being advantageously a pool of normalhuman plasmas.

As it will be shown hereinafter, the process of the invention isspecific and effective. It provides “intra-test” coefficients ofvariation of less than 3%, and “inter-test” coefficients of variation ofless than 5%. Moreover, it is insensitive to the rates of factors of theintrinsic pathway, to fractionated heparins of low molecular weight, tothe rates of PS, to the rates of factor VIII, to the CAC's, to the VKA'sand to the mutation 20210A of prothrombin.

The variant of this process, which is recommended in consideration ofits ease of practice, its lower cost and its suitability for being moreeasily automated on present-day machines, is an improved process.

According to this variant, therefore, an improved process is offered fordetermining APC-R in a test specimen of human plasma, the process, whichemploys the initiation of coagulation at the X factor by means of asnake venom in the presence of a mixture of Ca²⁺ and exogenic APC, andthen the measurement of the coagulation time (T), being characterized inthat it includes the steps consisting of:

(1a) bringing into contact

(a) a test specimen of human plasma

(b) a reactant deficient in factor V (VdfR) as a product providing most(preferably all) of the coagulation factors other than factor V, and

(c) CVH as a product specifically activating X to Xa, and incubating themixture thus obtained, for at least 1 minute at a temperature between 10and 45° C., preferably for 3 to 5 minutes at a temperature of 35 to 40°C., and better for 4 minutes at 37° C.;

(2a) introducing the mixture thus incubated Ca²⁺ and exogenic APC;

(3a) measuring the coagulation time (T) and

(4a) estimating APC-R by comparing the coagulation time (T) thusmeasured with the experimentally determined reference coagulation time(T₀) having statistically a value of about 110 s. T<T₀ indicates thatthe human plasma test specimen has a resistance to the activated proteinC, and T >T₀ indicates that the human plasma test specimen has noresistance to the activated protein C.

The operations set forth above for steps (1), (2, ii) and (3, ii) applyvery surely to steps (1a), (2a) and (3a) of the variant of the processof the invention.

From the practical point of view, T₀ has a length of 20 s (110±10 s).Thus, when T is greater than 120 s the plasma tested is normal, and whenT is less than 100 s the plasma tested it is certainly APC-R.

There is really an interval of doubt (in English: “grey zone”) whichcorresponds closely to the aforementioned interval [0.71≦N-APC-R-SR≦0.90of the principal process] when 100×≦T≦120 s. If the coagulation timemeasured is in the gray area of doubt (100 s≦T≦120 s), it is recommended(i) to employ the principal process by increasing the concentration ofthe plasma (as indicated above), or (ii) to do an MB study which willgive an unequivocal result.

The value T₀=110 s and especially the gray area 100 s≦T≦120 s have beendetermined experimentally on several lots of known human plasmas [normalplasmas (MB−) and APC-R (MB+) plasmas] by bringing into contact for 4minutes at 37° C. one volume of each plasma to be studied (previouslydiluted 1:10 in OKB), 1 volume of VdfP enriched with PL and 1 volume ofCVH, and introducing into the resultant mixture 1 volume of an APCsolution containing CaCl₂, and then measuring the time T. For furtherdetails see Examples 13 and 14 below.

Other advantages and features of the invention will be better understoodfrom the reading that follows of embodiments and tests. None of thesethings is restrictive but are given by way of explanation. For thereader's information, it is stated that Examples 1-3 concern obtainingvarious products and reactants involved according to the invention;examples 4-12 relate to the execution of the process of the invention,and examples 13-14 relate to variants of this process.

EXAMPLE 1

Obtaining the Human Plasma for Testing The plasma is drawn into an 0.102M solution of trisodium citrate, at the rate of 1 volume of citrate per9 volumes of blood. The specimen is then centrifuged at 3500 g for 15minutes at 4° C. It is recommended to perform a double centrifugation at4° C. so as to eliminate any platelets from the specimen. The separationand collection of the plasma must be performed as quickly as possibleafter drawing.

After decantation the plasma can be stored at

2-8° C. for 8 hours.

15-19° C. for 8 hours, and

−20° C. for 1 month.

For thawing, the plasma must be thawed at 37° C. in athermostat-controlled water bath for at least 10 minutes, thenhomogenized before use. Refreezing a plasma is to be ruled out.

EXAMPLE 2

Obtaining a Control Plasma

The procedure is as indicated in Example 1.

EXAMPLE 3

Preparation and Preservation of the Reactants

First Reactant (R1)

R1 is a lyophilized human plasma immunodepleted of factor V and enrichedwith PL. The lyophilized product is made into an aqueous compositionwith 2 ml of distilled water. The resultant solution is homogenized atthe ambient temperature (18-25° C.) before use.

This solution is stable at

18-25° C. for 6 h

15-19° C. (in the automatic analyzer sold under the trademark “STA” bythe Diagnostica Stago company) for 38 h

2-8° C. for 26 h, and

−20° C. for 1 month

Second Reactant (R2)

R2 is the lyophilizate of CVH. This lyophilized product is made into anaqueous composition with 2 ml of distilled water (which canadvantageously contain 1% v/v of PB). The resultant composition ishomogenized at the ambient temperature (18-25° C.) before use.

This composition is stable at:

18-25° C. for 6 h

15-19° C. in the STA analyzer for 38 h

2-8° C. for 26 h and

−20° C. for 1 month.

Third Reactant (R3)

R3 is the activated human protein C in a lyophilized calcium medium. Thelyophilizate is poured into 1 ml of distilled water. The resultantsolution is homobenized at the ambient temperature (18-25° C.) beforeuse.

This solution is stable at:

18-25° C. for 6 h

15-19° C. in the STA analyzer for 38 h,

2-8° C. for 26 h, and

−20° C. for 1 month.

EXAMPLE 4

Procedure

Identical volumes of the various products and reactants (unlessotherwise stated) which are obtained according to Examples 1-3 above)which are involved in the determination process according to theinvention. These volumes are between 5 μl and 50 μl. The specificationsthat follow are those relating to the measurements made with theabove-mentioned STA analyzer; the following are thus used:

50 μl of a test specimen of human plasma (or from a pool of normalplasmas), diluted 1:10 in OKB, 50 μl of R1 (i.e., VdfP enriched withPL), 50 μl of R2 (i.e., CHV) and 50 μl of CaCl₂ in aqueous solution orof R3 (i.e., APC+Ca²⁺).

In the STA analyzer the human test plasma or pool of normal plasmas, thePL-enriched VdtP, and CVH are combined. The resultant mixture isincubated for 4 minutes at 37° C.

The solution of CaCl₂ or the mixture of CaCl₂+APC (i.e., R3) isintroduced into the mixture after having preheated each to 37° C. TheSTA determines the coagulation time per sample tested and the controlpool in the presence and in the absence of APC.

The results obtained are directly expressed by the analyzer in the formof the above-mentioned standardized ratio: N-APC-R-SR.

EXAMPLE 5

Determination of the Normal Range

Starting with 30 pouches of plasma supplied by the Centre de TransfusionSanguine [blood transfusion center], the normal ratio standardized by anormal pool of 25 men and 25 women, as well as the pathological zone ina lot of 15 plasmas MB+ (homozygous and heterozygous APC-R plasmas). Itwas found that the normal value for the MB− plasmas is such thatN-APC-R-SR≧0.80 with the STA analyzer.

EXAMPLE 6

Repeatability within Tests

Starting with 2 APC-R homozygous plasmas (MB+), identified as APC-R 1and APC-R 2, and with a normal pool serving as control (MB−), 21determinations were made in triplicate. The results relating to thecoefficient of variation are given in the following Table I. They showthat the coefficient of variation of the standardized N-APC-R-SR ratiois less than 3%.

TABLE I Coefficient of variation of N-APC-R-SR Lot 005 Lot 006 Lot 007Pool MB- 2.08 1.94 2.25 APC-R 1 (MB+) 1.64 0.96 1.32 APC-R 2 (MB+) 1.601.82 1.57

EXAMPLE 7

Day-to-Day Repeatability

To estimate the day-to-day repeatability, a frozen normal plasma (MB−)and 2 pathological plasmas (MB+), one frozen and the other lyophilized,were divided into aliquots and then tested for 10 days with the same lotof reactants on the same STA analyzer. The results are shown in TableII. They show that the coefficient of the day-to-day variation ofN-APC-R-SR is less than 5%.

TABLE II MB + MB + frozen MB + lyophilized N-APC-R-SR N-APC-R-SRN-APC-R-SR M 0.93 0.51 0.56 SD 0.93 0.02 0.02 CV % 3.66 3.99 4.08 Notes:M: average of 10 tests per plasma SD: standard deviation from the meanCV %: Coefficient of variation

EXAMPLE 8

Repeatability Per Lot

The STA analyzer was used to test a normal plasma MB− and a pathologicalplasma MB+ (heterozygous APC-R plasma) which were divided into aliquotsand frozen. Immediately after thawing, each plasma was tested five timesin the same series (one flask), and 15 independent series (15 flasks ofthe same log) were performed. The results given in Table III show thatthe coefficient of variation is less than 5%.

TABLE III T or Normal Plasma (M−) Pathological Plasma (MB+) ratio M SDCV % M SD CV % T w/o  41.93 s 07.757 1.804 38.46 s 0.602 s 1.564 APC Twith 138.86 s  5.898 s 4.247 38.46 s 1.568 s 2.639 APC APC-R-  3.31 0.157 4.738  1.54 0.035 2.236 SR N-APC-  1.230  0.052 4.242  0.5750.010 1.807 R-SR (median) Notes: T: coagulation time (in seconds) M:average of the tests SD: standard deviation from the average CV:coefficient of variation (expressed in %)

EXAMPLE 9

Lot-by-Lot Repeatability

2 pathological plasmas identified as MB+1 and MB+2 (homozygous plasmas)and a normal plasma MB− were thawed and studied in 3 different lots ofthe same reactants (average of 5 measurements per test). A second normalplasma identical to the one before was also tested in parallel todetermine the ratio N-APC-R-SR. The results shown in Table IV show thatthe process of the invention is repeatable lot-by-lot.

TABLE IV Plasmas n-APC-R-SR tested Lot 005 Lot 006 Lot 007 Δ max MB −1.17 1.10 1.07 0.10 MB + 1 0.54 0.52 0.55 0.03 MB + 2 0.55 0.52 0.540.03 Note: Δ max: maximum variation

According to Table IV, Δ max (as absolute value) is less than or equalto 0.05 for plasmas MV+ and less than or equal to 0.10 for the plasmaMB−.

EXAMPLE 10

Influence of the Rate of Factor V

To highlight the specificity of the process of the invention in regardto the V factor, a series of dilutions of a normal pool or of an APC-Rplasma in a VdfP, on the one hand and a series of dilutions of the sameplasma APC-R in the normal pool, on the other.

The results obtained are given in FIGS. 1 and 2.

Curve A in FIG. 1 shows the variation of the standardized ratioN-APC-R-SR according to the concentration (in % v/v) of the pool ofnormal plasmas in the mixture (pool of normal plasmas)+(VdfP).

Curve B of the FIG. 1 shows the variation of the standardized ratioN-APC-R-SR with the concentration (in % v/v) of the plasma APC-R in themixture (plasma APC-R)+(VdfP).

Curve C in FIG. 2 shows the variation of the standardized ratioN-APC-R-SR with the concentration (in % v/v) of the same plasma APC-R inthe mixture (plasma APC-R)+(pool of normal plasmas).

Curves A and B show that:

with regard to the dilution of the plasma APC-R in a VdfP, a “positive”result (N-APC-R-SR≦0.71) when the plasma APC-R is in a concentration of10 to 100% v/v; and

with regard to the dilution of the normal plasma pool in a VdfP, a“negative” result (N-APC-R-SR≧0.98) when the normal plasma pool is in aconcentration of 10 to 100% v/v.

Consequently, curves A and B of FIG. 1 show clearly the specificity ofthe process of the invention with regard to Leiden's mutation of factorV.

This specificity is confirmed by curve C of FIG. 2 which shows that thedilution of the plasma APC-R in the pool of normal plasmas leads to a“positive” result (N-APC-R-SR>0.80) starting from a concentration of 20%v/v of plasma APC-R in the mixture 9APC-R)+(pool of normal plasmas).

EXAMPLE 11

Comparative Test 1 For comparison, 180 plasmas were tested in the STAanalyzer, viz. 60 MB− and 120 MB+ (60 homozygous and 60 heterozygous),identified as MB+ A and MB+ B, using as coagulation initiator eitherRVV-X according to the teaching of EP-A-0711838 [with a volumetric ratioVdfP/(VdfP+plasma under test)=75%], or CVH according to the invention[with a volumetric ratio VdfP/(VdfP+ plasma under test)=50%]. Theresults given in Table V show that the process of the invention givesbetter sensitivity. In this comparative test, a second pool of 50 normalMB− plasmas was used as control.

TABLE V Number of Plasmas Tested Plasmas Control by MB By the prior artBy the invention used + − + − + − MB −  0 60  0 60  0 60 MB + A 60  0 59 1 60  0 MB + B 60  0 57  3 60  0

EXAMPLE 12

Comparative Test 2

Taking inspiration from the teaching of WO-A-9604560 [viz. a singlevenom (RVV-X) for the activation of X to Xa], RVV-X was compared withCVH in the determination of APC-R. To this end, two pathologicalplasmas, identified as APC-R 1 and APC-R 2), diluted 1:10 in OKB, inrelation to a pool of normal plasmas, using furthermore two controls:one plasma MB− and one plasma MB+. The results obtained are given intable VI below show that CVH has a discrimination superior to that ofRVV-X. For the plasmas APC-R 1 and APC-R2, RVV-X gives a standardizedratio N-APC-R-SR that is within the gray area 0.72≦N-APC-R-SR≦0.90 (0.72for APC-R 1 and 0.77 for APC-R 2), whereas CVH gives a standardizedratio (0.57 for APC-R 1; 0.58 for APC-R 2) that is decidedly below theminimum (0.72) of the gray area.

TABLE VI Plasmas Pool MB − MB + normal control control APC-R 1 APC-R 2RVVX T (s) 36,6/79.9 42,3/86.8 40.2/52.4 43.4/63.2 45.0/70.6 ΔT (s) 40.344.5 12.2 19.8 25.6 rs 2.01 2.05 1.30 1.45 1.56 rn — 1.02 0.64 0.72 0.77CVH T (s) 39.3/14.4 43.8/118.0 42.1/71.1 43.0/71.9 46.0/78.0 ΔT (s) 75.174.2 29.0 28.9 32.0 rs 2.91 2.69 1.68 1.67 1.69 rn — 0.92 0.57 0.57 0.58Notes T: coagulation time (in seconds) without APC/with APC ΔT:variation of coagulation time T(with APC) - T(without APC) rs: APC-R-SRrn: N-APC-R-SR

EXAMPLE 13

Procedure

Identical volumes of the plasma to be studied and of the reactants R1,R2 and R3 obtained from examples 1 and 3 above, respectively. Thesevolumes are between 5 μl and 50 μl. The following procedures are thoserelating to the work performed by means of the STA analyzer referred toabove; thus the following were used:

50 μl of human plasma test specimen diluted 1:10 in OKB,

50 μl of R1 (i.e., PL-enriched VdfP)

50 μl of R2 (i.e., CHV) and

50 μl of R3 (i.e., APC+Ca²⁺)

In the STA analyzer, the human plasma to be tested, VdfP enriched withPL, and CVH are combined. The mixture is incubated for 4 minutes at 37°C.

R3 (i.e., CaCl₂+APC) , preheated to 37° C., is introduced into themixture thus obtained. The STA determines the coagulation time (T) forthe plasma sample being tested.

Depending on the result obtained, three cases present themselves:

if T>120 s, the plasma tested is not APC-R [it is a normal plasma(MB−)];

if 120 s≦T≦100 s, there is doubt (to clear the doubt, the procedureindicated above is followed, or for MB), and

if T<100 s, the plasma is an APC-R plasma (plasma MB+).

Table III above illustrates such a result, the coagulation time (withAPC) of the plasma MB− being 138.86 s, and that of the plasma MB+, 59.42s. The same is the case with Table VI in regard to the tests involvingCVH.

EXAMPLE 14

Effectiveness

Proceeding according to the directions given in Example 13, 582 humanplasmas were studied, divided into two lots and each analyzed by MB, lot1 comprising 398 plasmas (14 MB+ plasmas and 384 MB− plasmas), and lot 2comprising 184 plasmas (93 MB+ plasmas and 91 MB− plasmas). The resultsobtained are given in Table VII below, where each coagulation time (T)is stated for periods of 10 s (60 to 160 s) and beyond 160 s.

TABLE VII Plasmas Tested Coagulation Time Lot 1 (n = 398) Lot 2 (n =184) T (in seconds) (A) (B)  60 < T ≦ 70  5 MB+  5 MB+  70 < T ≦ 80  8MB+ 38 MB+  80 < T ≦ 90  1 MB+ 37 MB+  90 < T ≦ 100 0 12 MB+ 100 ≦ T ≦110 0  1 MB+ 110 < T ≦ 120  1 MB− 0 120 < T ≦ 130  5 MB− 0 130 < T ≦ 14042 MB−  2 MB− 140 < T ≦ 150 81 MB−  5 MB− 150 < T ≦ 160 86 MB−  7 MB− T< 160 167 MB−  77 MB− Notes A: Number and nature of the plasmas of lot 1that have coagulated B: Number and nature of the plasmas of lot 2 thathave coagulated

The results given in Table VII show that the determination techniqueused (i) gives no false positive and no false negative in lots 1 and 2(i.e., no MB− plasma is classed among the MB+ plasmas, and conversely noMB+ plasma is classified with the MB− plasmas), and (ii) gives only alimited number (<0.6%) of doubtful values [of the 582 plasmas tested,only 3 plasmas are within the gray area (100×≦T≦120 s), namely; 2 MB−plasmas in lot 1 and 1 MB+ plasma in lot 2].

What is claimed is:
 1. A process for determining a resistance toactivated protein C of a test specimen of human plasma comprising thesteps of: (1) mixing together (a) said test specimen of human plasma,(b) a reactant deficient in factor V which supplies at least most of thecoagulation factors other than factor V, and (c) venom of Crotalusviridis helleri which specifically activates factor X to Xa andincubating the resulting mixture of (a), (b) and (c) for at least oneminute at a temperature of between 10 and 45° C.; (2) introducing intothe incubated mixture(i) Ca²⁺ or (ii) Ca²⁺+exogenic activated protein C;(3) determining the coagulation time (i) in the absence of activatedprotein C and (ii) in the presence of activated protein C; (4) repeatingsteps (1) to (3) replacing, in step (1), said test specimen with anormal plasma as control; and (5) correlating resistance to activatedprotein C by comparing the determinations made in steps (3) for saidtest specimen and (4) for said normal plasma, wherein initiation ofcoagulation is by means of activating factor X to Xa by said venom inthe presence of (i) Ca²+ or (ii) Ca²⁺+exogenic activated protein C. 2.The process of claim 1, wherein said reactant deficient in factor V is ahuman or animal plasma deficient in factor V.
 3. The process of claim 1,further comprising the step of adding phospholipids to the mixture ofstep (1).
 4. The process of claim 1, wherein said test specimen of humanplasma of step (1) is obtained by treatment with trisodium citrate,centrifuged at 3500 g for 15 minutes and diluted with Owen-Kollerbuffer.
 5. The process of claim 1, wherein said normal plasma of step(4) is diluted under identical conditions to those of said test specimenof human plasma.
 6. The process of claim 1, further comprising making acomparison of a sensitivity to the activated protein C of said testspecimen and said control plasma, said sensitivity being a ratio, calledAPC-R-SR, of the coagulation time in the presence of the activatedprotein C (APC) to the coagulation time in the absence of the activatedprotein C:${{APC}\text{-}R\text{-}{SR}} = \frac{{coagulation}\quad {time}\quad {in}\quad {presence}\quad {of}\quad {APC}}{{coagulation}\quad {time}\quad {in}\quad {absence}\quad {of}\quad {APC}}$

and normalizing the correlation made in step (5) by a standardizedratio, called n-APC-R-SR, of the sensitivity to activated protein C ofsaid test specimen to the sensitivity to activated protein C of thenormal control plasma:${n\text{-}{APC}\text{-}R\text{-}{SR}} = {\frac{\text{(}{APC}\text{-}R\text{-}{SR}\text{)}_{specimen}}{\text{(}{APC}\text{-}R\text{-}{SR}\text{)}_{{control}\quad {plasma}}}.}$


7. The process of claim 6, wherein a standardized ratio wheren-APC-R-RS>0.80 generally indicates that said test specimen has noresistance to the activated protein C, and a standardized ratio wheren-APC-R-SR<0.80 generally indicates that said test specimen does haveresistance to the activated protein C.
 8. The process of claim 1,wherein the normal plasma used as the control is a pool of normal humanplasmas.
 9. The process of claim 1, wherein in step (1), said testspecimen is diluted to a concentration greater than or equal to 1:20 v/vand said reactant is a human plasma deficient in factor V enriched withphospholipids and said mixture is incubated for 4 minutes at 37° C. 10.The process of claim 9, wherein said test specimen is diluted withOwen-Koller buffer.
 11. The process of claim 1, wherein in step (4),said normal plasma is a pool of normal human plasmas.
 12. The process ofclaim 1; further comprising normalizing of the correlation of resistanceto activated protein C by comparing the coagulation time (T) obtained instep (3, ii) with a reference coagulation time (T₀) determined byexperiment, where T<T₀ generally indicates that the test specimen ofhuman plasma has resistance to the activated protein C, and T>T₀generally indicates that the test specimen of human plasma has noresistance to the activated protein C.
 13. The process of claim 12,wherein T₀ has a value of about 110 seconds.
 14. The process of claim12, wherein T₀ is 110±10 s, whereby: when T>120 s, said test specimenhas no resistance to the activated protein C, and when T<100 s, saidtest specimen does have resistance to the activated protein C, aninterval between 100s≦T≦120 s being the interval of doubt.
 15. A processfor determining resistance to activated protein C of a test sample ofhuman plasma, comprising the steps of: (1) mixing together (a) said testspecimen of human plasma, (b) a reactant deficient in factor V whichprovides at least most of coagulation factors other than factor V, and(c) venom of Crotalus viridis helleri which specifically activatesfactor X to Xa, and incubating the resulting mixture of (a), (b) and (c)for one minute at a temperature of between 10 and 45° C.; (2)introducing Ca²⁺ and exogenic activated protein C into said mixture ofstep (1); (3) measuring a coagulation time (T); and (4) correlatingresistance to activated protein C by comparing the measured coagulationtime (T) with an experimentally determined reference coagulation time(T₀) having a value of about 110 seconds, where T<T₀ indicates that saidtest specimen has a resistance to activated protein C, and T>T₀indicates that said test specimen has no resistance to activated proteinC, wherein initiation of coagulation is by means of activating factor Xto Xa by said venom in the presence of a mixture of Ca²⁺ and exogenicAPC.
 16. The process of claim 15, wherein T₀ is 110 ±10 s, whereby: whenT>120 s, said test specimen has no resistance to the activated proteinC, and when T<100 s, said test specimen does have resistance to theactivated protein C, an interval between 100s ≦T≦120 s being theinterval of doubt.
 17. The process of claim 15, wherein said mixture ofstep (1) is incubated for 3 to 5 minutes at a temperature of between 35to 40° C.
 18. The process of claim 15, wherein said mixture of step (1)is incubated for 4 minutes at a temperature of 370° C.